Oxidative Stress Biomarkers in Plasma

The antioxidant barrier and the concentration of Reactive Oxygen Metabolites (ROMs) were measured using the OXY-Adsorbent (2 μL of plasma) and d-ROMs tests (5 μL of plasma, DIACRON INTERNATIONAL, s.r.l, Italy) following the manufacturer’s protocol (for detailed description of these tests, see [39 (link)]). The OXY adsorbent test was used to quantify the ability of the plasma antioxidant barrier to buffer massive oxidation through hypochlorous acid, while the d-ROMs test mostly measures hydroperoxydes as a marker of global early oxidative damage (principally on lipids and proteins). Antioxidant barrier is expressed as mM of HClO neutralised and d-ROMs as mg of H₂O₂ equivalent/dL. All measurements were run in duplicates and intra-individual variation was low (respectively 1.96 ± 0.34% for the OXY test and 2.76 ± 0.86% for the d-ROMs test). Measurements for the same individual before and after reproduction were run within the same laboratory session, and measurements of all the samples were divided in three laboratory sessions. Inter-session variations in the measurement (based on one sample repeated in all the session) were 4.52% for the OXY test and 5.31% for the d-ROMs test. Repeatability, i.e. the proportion of variability explained by the individual, was calculated following [40 (link)]. Both d-ROMs (ANOVA, F_{1, 35} = 26.41, p <0.001, r = 0.585) and Oxy-Adsorbent (ANOVA, F_{1, 35} = 4.90, p <0.034, r = 0.178) tests were shown to be repeatable over the study.